12 bp barcoded primers

After being removed from the sampler, the stool was diluted 5X with molecular grade water and homogenized with a vortex. The stool suspension (250 µl) was used for DNA extraction. The genomic DNA was purified and isolated with MagaBio Soil/Feces Genomic DNA Purification Kit (Hangzhou Bioer Technology, Hangzhou, Zhejiang, China). The extracted DNA was quantified at an A260/A280 nm ratio with the NanoDrop ND-1000 (Thermo Fisher Scientific, Cleveland, OH, USA). Then extraction from each sample was diluted to approximately 5 ng/µl and stored at – 20 °C for 16S rRNA sequencing.
The 12-bp barcoded primers synthesized by Invitrogen (Invitrogen, Carlsbad, CA, USA) were used to amplify the bacterial 16S rRNAV3-V4 fragments. The PCR mixture contained 25 μl reactions Taq (Takara Biotechnology, Dalian, Liaoning, China), 1 μl of each primer (10 mM), and 3 μl DNA (20 ng/μl) template (final volume: 50 μl). The PCR protocol was as follows: 94 °C for 30 s followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 30 s; followed by a final extension at 72 °C for 10 min. The PCR products were subjected to 1% agarose gel electrophoresis and then sequenced on an Illumina Miseq (PE 300) in the MAGIGENE Genomic Institute. The PCR products were mixed according to the Instructions of the GeneTools Analysis Software (Version 4.03.05.0, SynGene).

After being removed from the sampler, the stool was diluted 5X with molecular grade water and homogenized with a vortex. The stool suspension (250 µl) was used for DNA extraction. The genomic DNA was purified and isolated with MagaBio Soil/Feces Genomic DNA Purification Kit (Hangzhou Bioer Technology, Hangzhou, Zhejiang, China). The extracted DNA was quantified at an A260/A280 nm ratio with the NanoDrop ND-1000 (Thermo Fisher Scientific, Cleveland, OH, USA). Then extraction from each sample was diluted to approximately 5 ng/µl and stored at – 20 °C for 16S rRNA sequencing.
The 12-bp barcoded primers synthesized by Invitrogen (Invitrogen, Carlsbad, CA, USA) were used to amplify the bacterial 16S rRNAV3-V4 fragments. The PCR mixture contained 25 μl reactions Taq (Takara Biotechnology, Dalian, Liaoning, China), 1 μl of each primer (10 mM), and 3 μl DNA (20 ng/μl) template (final volume: 50 μl). The PCR protocol was as follows: 94 °C for 30 s followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 30 s; followed by a final extension at 72 °C for 10 min. The PCR products were subjected to 1% agarose gel electrophoresis and then sequenced on an Illumina Miseq (PE 300) in the MAGIGENE Genomic Institute. The PCR products were mixed according to the Instructions of the GeneTools Analysis Software (Version 4.03.05.0, SynGene).